In Vitro PIG-A Gene Mutation Assay in Human B-Lymphoblastoid TK6 Cells

Chang-Hui Zhou,Chun-Rong Yu,Ze-Hao Zhao,Ruo-Wan Li,Jing-Ting Wang,Peng-Cheng Huang,Jing Ma,Yan Chang,Tian-Tian Zhao

doi:10.1055/s-0041-1735146

Abstract

AbstractThe X-linked PIG-A gene is involved in the biosynthesis of glycosylphosphatidylinositol (GPI) anchors. PIG-A mutant cells fail to synthesize GPI and to express GPI-anchored protein markers (e.g., CD59 and CD55). In recent years, in vitro PIG-A assay has been established based on the high conservation of PIG-A/Pig-a loci among different species and the large data from the in vivo system. The purpose of this study was to extend the approach for PIG-A mutation assessment to in vitro human B-lymphoblastoid TK6 cells by detecting the loss of GPI-linked CD55 and CD59 proteins. TK6 cells were treated with three mutagens 7,12-dimethylbenz[a]anthracene (DMBA), N-ethyl-N-nitrosourea (ENU), etoposide (ETO), and two nonmutagens: cadmium chloride (CdCl2) and sodium chloride (NaCl). The mutation rate of PIG-A gene within TK6 cells was determined on the 11th day with flow cytometry analysis for the negative frequencies of CD55 and CD59. The antibodies used in this production were APC mouse-anti-human CD19 antibody, PE mouse anti-human CD55 antibody, PE mouse anti-human CD59 antibody, and nucleic acid dye 7-AAD. An immunolabeling method was used to reduce the high spontaneous level of preexisting PIG-A mutant cells. Our data suggested that DMBA-, ENU-, and ETO-induced mutation frequency of PIG-A gene was increased by twofold compared with the negative control, and the effects were dose-dependent. However, CdCl2 and NaCl did not significantly increase the mutation frequency of PIG-A gene, with a high cytotoxicity at a dose of 10 mmol/L. Our study suggested that the novel in vitro PIG-A gene mutation assay within TK6 cells may represent a complement of the present in vivo Pig-a assay, and may provide guidance for their potential use in genotoxicity even in cells with a significant deficiency of GPI anchor.

Highlights

Genotoxicity tests are utilized to identify compounds with a potential risk for carcinogenicity and heritable mutations,# These authors contributed to this work.which require a battery of tests to cover different genetic endpoints, such as DNA damage, gene mutation, as well as structural or numerical chromosomal abbreviation.[1]
Our study suggested that the novel in vitro PIG-A gene mutation assay within TK6 cells may represent a complement of the present in vivo Pig-a assay, and may provide guidance for their potential use in genotoxicity even in cells with a significant deficiency of GPI anchor
The aim of this study was to develop an in vitro PIG-A gene mutation assay in TK6 cells via detection of the loss of GPIlinked CD55 and CD59 proteins by flow cytometry

Summary

Introduction

Genotoxicity tests are utilized to identify compounds with a potential risk for carcinogenicity and heritable mutations,# These authors contributed to this work.which require a battery of tests to cover different genetic endpoints, such as DNA damage, gene mutation, as well as structural or numerical chromosomal abbreviation.[1]. Genotoxicity tests are utilized to identify compounds with a potential risk for carcinogenicity and heritable mutations,. E78 In Vitro PIG-A Gene Mutation Assay in Human B-Lymphoblastoid TK6 Cells Zhou et al. Mutation assay for a rodent-based endogenous phosphatidylinositol class A gene (PIG-A in humans and Pig-a in rodents) has become a recognized method for detecting potential mutagenicity of exogenous compounds.[2] The PIG-A/Pig-a gene is involved in early synthesis of cell-surface glycosylphosphatidylinositol (GPI) anchors, which was usually employed by GPI anchor proteins (e.g., CD59, CD55, or CD90) to bind to the cell membrane. In each cell, there is only one copy of the functional PIG-A/Pig-a gene, and a single inactivating mutation in this gene will result in the inactivation of the enzymatic function of the PIG-A protein and the subsequent deficiency of all GPI-linked surface proteins.[3,4]

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